Supplementary information for Nitrogen cycle feedbacks as a control on euxinia in the mid-proterozoic ocean, by R.A.Boyle, J.R.Clark, G.

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1 Supplementary information for Nitrogen cycle feedbacks as a control on euxinia in the mid-proterozoic ocean, by R.A.Boyle, J.R.Clark, G. Shields-Zhou, S.W.Poulton, D.E. Caneld & T.M.Lenton. Nature Communications

2 Supplementary gures Supplementary gure S1 - Schematic of Caneld's 2006 ve box model of an example coastal upwelling zone. The surface ocean is represented by two boxes - a coastal upwelling region photic zone U, and an outer-ocean surface box S away from the inuence of upwelling. There are two intermediate depth boxes - The upwelling region subphotic zone U M (the continental shelf below the photic zone, into which production sinks, where oxygen depletion potentially happens, and the composition of which is the main focus of our arguments) and the outer ocean subphotic zone I. The non-coastal upwelling boxes S and I are inactive in the sense that their biogeochemical uxes are not explicitly resolved and their purpose in the model is to act as concentration buers to the upwelling zone photic U and subphotic U M boxes. Nutrient upwelling also enters the upwelling region from the wider deep ocean box D. The mixing constants K j represent a combination of vertical and horizontal diusion - the former over scales of m, the latter potentially over the scales of 100's of kilometres over which transport occurs on isopycnal surfaces. Each diusion constant is thus the product of a horizontal diussion coecient K and the hieght H and length L scale over which diussion occurs (e.g. K I = HK HO L cmh 1 ), see Caneld 2006 for further 2 details. Advective ow occurs into the UM box at rate Acmh 1 from the deep ocean box D and at rate Bcmh 1 from intermediate depths I. In the original model export production sinking in to the UM box is written as a given fraction P artu of total production a fraction x of which is degraded in the UM box and 1 x escapes to the deeper ocean box D for burial or respiration there. (In this version we neglect this deep ocean export production, so that in the terminology of the model description below export production EP = xp artu, x = 1). The main modication from the original model is the explicit separation of export production into a component based on assimilatory nitrate reduction EP Nox and one based on direct ammonium assimilation EP NR. The respiratory uxes on which we focus involve the electron acceptors oxygen R aerobic, nitrate R denif and sulphate R SR, which collectively account for complete breakdown of export production under most of the parameter choices shown. Further details are given in the main text. 2

3 Sensitivity analysis to oxygen and ammonium concentrations A. B. C. D. E. F. Supplementary gure S2 - Upwelling zone composition at O 2U = 200µM This plot and those in the remainder of this subsection are a repeat of the results in the main paper using a wider range of values for two key parameters; (i) the amount of oxygen in the surface ocean O 2U (supplementary gures S2-S4), (ii) the steady deep/intermediate depth ammonium concentration N RI, N RD in the non-xation high reduced nitrogen scenario (supplementary gures S5-S7). As for gure 3 in the main text, the top row (sub gures A-C) of each gure gives the various nutrient concentrations, and the bottom row (sub gures D-F) the magnitude of the various uxes. The three main cases are shown in each column, the non-xation (left), xation (centre) and non-xation with a standing deep ocean ammonium pool (right). Ammonium scenarios include N RI = 1µM, N RD = 1µM (in addition to any ammonium released from anaerobic remineralisation). Recall that non-zero export production only occurs in the xation case when nitrate concentration in the photic zone is in decit compared to phosphate. The following plots show a range of oxygen concentrations in the photic zone, deviating from the prescribed present day value O 2U = 250µM and in keeping with the high uncertainty concerning the oxygen level during the boring billion. Note that changes in the oxygen concentration of the upwelling zone do not alter the separation between the xation and non-xation states with respect to euxinia. As might logically be expected, the steady state oxygen concentration in the photic zone (O 2U ) and at intermediate depths (O 2I ) dictates the aerobic-anaerobic aportioning of the respiration of organic carbon. But although the quantitative value of the various respiratory uxes is aected by the oxygen concentration, the qualitative separation between the xation and non-xation cases, the absence of sulphate reduction in the former case, and all the arguments in the main text that stem from this separation, still hold across the range of oxygen parameterisations shown. Even at modern oxygen concentrations in the upwelling zone, the absence of oxygen in the deep ocean means the upwelling zone goes anoxic at realistic upwelling rates. Euxinia remains conned to the xation case. In the ammonium case (which only tracks ammonium-limited production) there is little to respire anaerobically, because ammonium is removed by nitrication due to the high oxygen concentration (which would in reality lead to increased nitrate-limited production). 3

4 A. A. B. C. D. E. F. Supplementary gure S3 - Upwelling zone composition for O 2U = 20µM A reduction in oxygen concentration to that equivalent 0.1P AL removes the nitrication sink for ammonium (right), and supports signicant ammonium-driven export production, leading to sulphate reduction lower down the water column, in the absence of nitrogen xation (i.e. in contrast to our arguments), provided that ammonium pool is stable (i.e. does not get nitried), and does not lead to build up of a nitrate pool. 4

5 A. B. C. D. E. F. Supplementary gure S4 - Upwelling zone composition for O 2U = 100µM and O 2I = 100µM Oxygenation of intermediate depths as well as the surface (i.e. the Precambrian-Cambrian boundary) to approximately half of present level would render our arguments irrelevant, because there would be sucient oxygen to respire export production. 5

6 A. B. C. D. E. F. Supplementary gure S5- Upwelling zone composition for N RI = N RD = 0µM In the absence of deep ocean ammonium the separation between the nitrate production/denitrication and xation/euxinic states is at its strongest. Note oxygen goes down in the ammonium case due to respiration of nitrate-driven production which is not tracked. 6

7 A. B. C. D. E. F. Supplementary gure S6- Upwelling zone composition for N RI = N RD = 0.5µM At low but non-zero ammonium concentrations the arguments for the previous supplementary gure still apply. 7

8 A. B. C. D. E. F. Supplementary gure S7- Upwelling zone composition for N RI = N RD = 2µM At high ammonium concentrations euxinia is relatively easy to achieve in all cases, because the feedback between nitrate concentration and export production is weakened. However, as we discuss in the main text, a large deep ocean ammonium pool in insucient as a long-term global ocean average - because its occurrence is contingent on nitrogen xation (therefore is encompassed by the xation case in the main results), or would be depleted by nitrication and lead to a reversion to the nitrate non-xation case. 8

9 Imperfections in the isotope estimate A. B. C. D. Supplementary gure S8 - No ammonium Dierent components of the isotope signal for the nitrate-based nitrogen cycle scenario. N oxi = 40, N oxd = 36, N RI = 0, N I = 0, O 2U = 100 (~0.5PAL), O 2I = 0, O 2D = 0. This plot and those below show a breakdown of the nitrogen isotope approximation, with and without the extra ammonium assimilation signal described in the text. Top left plot shows the δ approximation for upwelling zone nitrate in the xation (blue) and non-xation (green) cases, both with (circles) and without (solid lines) the inclusion of the ammonium isotopic composition in the nitrication signal. Top right plot shows the natural log of the upwelling zone F e 2+ /H 2 S ratio for the xation (blue) and non-xation (green) cases, relative to the pyrite deposition stoichiometry (dashed cyan line). Lower left plot shows a breakdown of the dierent components of the δ signal in the non-xation case (in the xation case nitrication of remineralised ammonium is assumed to be the only signicant signal). The default components of this signal (used for the graphs described in the main text) are given in yellow - denitrication (circles), nitrication (dashed line), nitrate assimilation (solid line), the modied nitrication signal impacted upon by ammonium assimilation is given in magenta. Lower right plot shows a breakdown of the steady state chemical composition of the upwelling zone, with the concentration of oxygen (blue), nitrate (green), ammonium (red) and hydrogen sulphide (cyan) given for the xation (dashed lines) and non-xation (solid lines) cases. Naturally, for a given component of the signal to be non zero, the ux to which that component corresponds must be non quantitative (i.e. 0 < f < 1 for f denif, f ammon, f NO and f ammon above), and the steady state concentration of 3 assim both reactant and product must be non-zero. Although ammonium assimilation (and numerous other fractionations that we neglect in our simplistic approach) makes interpretation of the δ record more complex, it does not alter our basic argument for a dissociation between a genuine positive denitrication δ signal and the evidence for euxinia during the Proterozoic. 9

10 A. B. B. C. D. D. Supplementary gure S9 - Some ammonium N oxi = 0, N oxd = 0, N RI = 1, N I = 1 In the absence of nitrate the denitrication isotope signal does not occur and the non-xation signal is dominated by the other components. 10

11 A. B. C. D. Supplementary gure S10 - High ammonium N oxi = 0, N oxd = 0, N RI = 2, N I = 2 Raising the ammonium concentration leads to increased alteration of the nitrication part of the signal by the isotopic composition of the ammonium entering the process. 11

12 A. B. C. D. Supplementary gure S11 - Mixed scenario N oxi = 40, N oxd = 36, N RI = 1, N I = 1, exceptionally -enriched ammonium 14 N N R0 = N standard. Even a highly -enriched ammonium pool and its subsequent nitrication cannot recreate the high positive enrichment of (residual) water column nitrate caused by denitrication. This denitrication signal remains separated in parameter space from euxinia, even in the presence of ammonium. 12

13 Supplementary table 1. and parameters References and denitions for model variables Quantity Description Default Value/Units Reference F e RD Concentration of 50µmolL 1 Holland, 1984 reactive F e(ii) in deep ocean box under ferruginous conditions F e RI Reactive F e(ii) at 50µmolL 1 intermediate depths F e RU Reactive F e(ii) at 0µmolL 1 surface N RD Deep ocean reduced 0µmolL 1 By hypothesis Nitrogen NH 4 + N RI Intermediate depths reduced Nitrogen NH 4 + 0µmolL 1 (1µmolL 1 in ammonium-driven non-xation case) N RU Surface reduced Nitrogen NH + 4 0µmolL 1 SO 4U Surface sulphate 2800µmolL present day, by hypothesis SO 4I Intermediate depth 2800µmolL 1 sulphate SO 4D Deep ocean sulphate 2800µmolL 1 O 2U O 2I O 2D Surface ocean oxygen (default value only) Intermediate depth oxygen Deep ocean oxygen (default value, subject to sensitivity analysis, see text) µmolL present day, by hypothesis 0µmolL 1 By hypothesis 0µmolL 1 P U Surface phosphate 0 when limiting to production, otherwise model variable (see text) (µmoll 1 ) P I Intermediate depth 0.1µmolL 1 ) phosphate P D Deep ocean phosphate 0.1µmolL 1 N oxu Oxidised nitrogen N ox = NO3 + NO 2, surface ocean (dominated by nitrate NO3 by hypothesis) 0µmolL 1 (unless a model output to assess stability of the xation state (see text) 13

14 Quantity Description Value/Units Reference Exportprod R aerobic R denif R SR K U K UM K I A B r C:P r N:P Flux of organic carbon sinking in to, and potentially available for respiration within, the upwelling zone Rate of aerobic respiration of organic carbon Rate of respiration of organic carbon via dissimilatory nitrate reduction Rate of respiration of organic carbon via dissimilatory sulphate reduction Vertical water exchange (diusive), between U and UM Vertical water exchange (diusive), between UM and D Horizontal water exchange (diusive) Advective upwelling from deep ocean to UM box Advective ow from intermediate depths into UM box organic matter redeld ratio parameter carbon to phosphorous organic matter redeld ratio parameter nitrogen to phosphorous r O2R :C org ratio between O 2 consumed and carbon oxidised during aerobic respiration of organic matter at Redeld stoichiometry model variable Caneld nmolc/cm 2 h 1 model variable (nmolc/cm 2 h 1 ) model variable (nmolc/cm 2 h 1 ) model variable (nmolc/cm 2 h 1 ) 0.1cm 2 h 1 Caneld cm 2 h 1 Caneld cm 2 h 1 Caneld model parameter, Caneld default value 0 (cm 2 h 1 ) model parameter Caneld Redeld, Redeld, /117 Anderson & Sarmiento,

15 Quantity Description Value/Units Reference r N:Corg organic matter nitrogen to carbon Redeld ratio 16/117 Redeld, 1958,. r NO3R :C org ratio between NO3 reduced and organic carbon oxidised during dissimilatory nitrate reduction 5 2 C 6H 12 O NO3 6N CO OH + 9H 2 O r SO4R :C org N oxd N oxi r a ratio between SO4 2 reduced and organic carbon oxidised during dissimilatory sulphate reduction CH 3 COO + SO4 2 + H 2 O H 2 S +2HCO3 +OH Deep ocean oxidised nitrogen Intermediate depth oxidised nitrogen Ratio between O 2 used and NO3 produced by subsequent nitrication during remineralisation of organic matter during oxic conditions 4/5 Konhauser / µmolL 1 (10µmolL 1 for gure in main text, by hypothesis) 40.43µmolL 1 (10µmolL 1 for gure in main text, by hypothesis) Caneld Caneld 2006, Anderson & Sarmiento 1994, (empirically derived approximation for organic matter approximately in Redeld stoichiometry) 15

16 Supplementary methods We relate the sulphate reduction ux to sulphide production by incorporating the possibility of sulphate limitation, which, although unlikely today could conceivably have occurred if the proterozoic global sulphate reservoir was towards the lower end of some estimates (e.g. Kah et al, 2004). Writing a sulphate mass balance for the upwelling zone, we get the sulphate concentration: SO 4UM = (A + K UM )SO 4D + (K I + B)SO 4I + K U SO 4U r SO4R :C org R SR A + B + K UM + K I From which we use the denominator to incorporate the possibility of sulphate limitation of dissimilatory sulphate reduction, if (S1) gives SO 4UM 0: R SRSO4 limited = 1 r SO4R :C org ((A + K UM )SO 4D + (K I + B)SO 4I + K U SO 4U ) Having incorporated this clause we get an approximation of H 2 S production (in the vicinity of organic carbon in the water column and surface sediments -i.e. the sort of context from which the empirical signatures originate) just by putting H 2 S r SO4R :C org R SR. Finally, we write a mass balance for reactive ferrous F e R, assuming that it is absent from the oxygenated surface box, solve for iron concentration and divide through by sulphide concentration to get a model estimate of the F e : H 2 S ratio: F e R : H 2 S = (A + K UM )F e RD + (K I + B)F e RI (A + B + K U + K UM + K I ) 1 r SO4R :C org R SR As discussed in the methods section in the nitrogen xation case we determine reduced nitrogen concentration directly, and in the non-xation state we assume that upwelling zone phosphate is in Redeld stoichiometry with nitrate P UMnofix = Nox UM +N R UM r N:P. We use a check to ensure that the nitrogen xation state remains valid, by requiring that for non-zero new production in the xation state there cannot be a build up of nitrate in the photic zone. Writing a mass balance for oxidised nitrate in the U box, and solving for its concentration: (S1) (S2) (S3) N oxufix = N ox I (A + B) + N oxum (A + B + K U ) EP r N:Corg A + B + K U Because nitrogen xation is only competitively viable when photic zone nitrate is decient relative to phosphate, we need to also consider the phosphate concentration of the photic zone. However, we have assumed that there will be no freely available phosphate in the photic zone during the xation state (because it will feed straight in to new production), therefore we approximate phosphate by the amount moving in from the upwelling zone, minus the amount absorbed into new production: P Ufix (A + B + K U )P UM EP r C:P We only consider the nitrogen xation state valid when N oxufix r N:P P Ufix > 0. This avoids (for example) solutions in which production, aerobic respiration and oxygen are all high in the nitrogen xation state, but this would realistically result in nitrication and the upwelling of stoichiometrically large quantities of NO3 to the photic zone which would ultimately undermine that state's existence. (S4) (S5) Estimating the organic carbon δ signal We couple our results to an approximate prediction of the kerogen δ signal. In the modern ocean organisms will record the δ signature of NO3, because this is the dominant form in which organic N is assimilated, an assumption that we retain here, based on the arguments about the transient nature of signicant NH 4 + build up. Because more energy is required to break chemical bonds containing than those containing 14 N, most reactions in the nitrogen cycle favour the lighter isotope. By convention this isotopic bias is measured relative to a standard: δ = 1000( 14 N sample 14 N standard 14 N standard ) 16

17 Where 14 N standard = (e.g. Robinson, 2001) is the isotope ratio within the atmospheric N 2 pool. A second useful metric is the change ε in the δ signal between a designated source and sink after a given reaction: ε = δ source δ sink 1 + δ sink 1000 = 1000( 14 N source 14 N sink 14 N sink ) δ source δ sink ε is therefore the dierence in δ between a substrate and its immediate product (for example, nitrogen xation favours 14 N via a value of ε , which by (S7) corresponds to a decline in the 14 N ratio of the xed organic nitrogen of around ). If an entire pool of N ows through a given reaction there is no change in fractionation. Consequently for a given reaction it is necessary to determine the fraction f of an initial substrate that is converted to a given product (using the formulation of, and ɛ values from, Robinson 2001, referenced in the main text): 14 N org = 15 N 14 N N2 (S6) (S7) δ substrate = δ 0substrate + ε reaction ln(1 f) δ product = ε reaction(1 f)ln(1 f) f The ocean nitrate reservoir is, of course, the sink with respect to N 2 xation ε fix = , and the source with respect to denitrication ε denif = and assimilatory nitrate reduction ε NO = 13 3 assim 1000 (Robinson et al 2001). For marine nitrication we use a conservative fractionation estimate ɛ nitrif = 15 (Robinson, 2001). For denitrication and nitrate assimilation, water column nitrate is, of course, the substrate, whereas for nitrication (and xation) is the product. Assuming a neutral default state of δ 0 = 0, using (S6) to (S9) give an expression for the change in isotopic fractionation in the NO3 reservoir of the upwelling zone: (S8) (S9) δ 15 (1 f fix )ln(1 f fix ) (1 f nitrif )ln(1 f nitrif ) N NO3 = ε fix +ε nitrif ε denif ln(1 f denif ) ε f NO ln(1 f fix 3 NO ) assim 3 assim f nitrif Assuming that a negligable fraction of the global N 2 reservoir goes through the biological nitrogen xation reaction f fix 0, consistent with the minimal impact of xation itself on fractionation (organic matter in N 2 -xing organisms has a δ close to zero) in comparison to the other uxes. It is necessary to make approximations to the fraction of upwelling zone nitrate going through denitrication f denif and assimilatory nitrate reduction f NO 3 assim, as well as the fraction of upwelling zone ammonium going through nitrication f nitrif. There is a clear dierence between the presence and absence of photic zone nitrogen xation. We make guesses at these fractions using the steady state uxes/concentration solutions above (note that this is the step at which signicant uncertainty is introduced, rendering our δ semi-quantitative): (S10) f deniffix = 0, f denifnofix = R denif r NO 3R :Corg N oxum f NO 3 assimfix = 0, f NO 3 assimnofix = EP N ox EP f nitrif = R aerobicr N:Corg N RUM Where we have assumed that all aerobically respired organic nitrogen is subsequently nitried, and that there is no denitrication or nitrate assimilation in the non N 2 xation state. By (S7), using the atmospheric standard as the main sink for NO3 leaving the marine reservoir 14 N standard = 14 N, we get the isotope ratio for upwelling sink zone nitrate: (S11) (S12) (S13) 17

18 ( 14 N ) NO 3 = ( ε NO ) 14 N standard ε Substituting (S6) into (S7) we note that we can simplify all this to δ = 1000( ( NO ) 14 N standard 14 N standard ) = 14 N standard ε NO, and therefore estimate the marine nitrate isotopic signature using the various fractionation terms above via 3 (S10): (S14) N RUM δ oxumfix = ε nitrif ( R aerobic r N:Corg 1)ln(1 R aerobicr N:Corg N RUM ) (S15) N RUM δ oxumnofix = ε nitrif ( R aerobic r N:Corg 1)ln(1 R aerobicr N:Corg N RUM ) ε denif ln(1 R denif r NO 3R :Corg ) N oxum ε NO ln(1 Exportprod N ox 3 assim Exportprod ) (S16) Equations (S15) and (S16) give the δ signal derived from upwelling zone NO3 for the steady states we have derived. The isotopic composition of ammonium available to undergo nitrication will be impacted upon by fractionations associated with ammonium assimilation, particularly in parts of parameter space in which a high fraction of export production is based on direct ammonium assimilation EP N R EP 1, with potential implications for the δ signal that we postulate. For the purposes of the simplistic treatment we aim for in this paper (i.e. for the arguments in the main text concerning the δ of nitrate-equilibrated material in the upwelling zone), the isotopic composition of the reduced nitrogen pool is relevant because it may inuence the isotopic composition of the nitrate produced by nitrication of that ammonium. ε nitrif = ( 14 N NO 3 14 N NH + 4 1) (Robinson, 2001). It is clear that if other processes alter the isotopic composition of ammonium sinking into the upwelling zone then this could signicantly impact the nitrication signal. In the photic zone the relevant processes are, of course, ammonium assimilation (increasing the -enrichment of residual non-assimilated ammonium N RU ) and decomposition of organic matter (reducing the 14 N by preferential 14 N mobilisation). We neglect any impact of NH 3 volatilisation because we do not envisage an a priori dierence between the xation and non-xation cases, and because the ux of NH 3 from the sea to the atmosphere is roughly equal to the deposition of NH 3 /NH 4 + on the sea surface today (Schlesinger & Hartley, 1992). An approximation of the isotopic composition of ammonium in the upwelling zone might be: δ RUM = δ RUM0 ε ammon (1 f ammon )ln(1 f ammon ) f ammon Where the left hand term is the (prescribed) initial isotopic composition of the ammonium pool in the upwelling zone, which can potentially altered by ammonication of organic nitrogen, which tends to preferentially degrade 14 N in organic matter, leaving behind relatively -enriched organic N (Sigman et al, 2009), according to ɛ ammon 5. The fraction f ammon of organic matter remineralised into ammonium (rather than feeding straight into nitrication) is set as the anaerobic fraction of respiration: f ammon = R denif + R SR R total Using the above approach we can also estimate the isotope ratio of reduced nitrogen in the upwelling zone, 14 N NRUM δ RUM = ( 1) 1000, so that: 14 N standard (S17) (S18) (S19) 18

19 = (1 + δ RUM ) 14 N N 14 RUM N standard 1000 We use this ratio to normalise the nitrication fractionation. We use a modern day estimate of the isotopic ratio of marine ammonium 14 N + NH 4 14 N standard δ NH + = 2.5 = 1000( )(Wada et al, 1975) 4 14 N N R0 = N. We are interested standard 14 N standard in the possibility that a dierent ammonium biogeochemistry in the distant past may have altered the isotope fractionation associated with nitrication, by dividing the baseline isotope ratio from the model (given in (S17)) by a normalised ammonium fractionation factor using (S20) scaled by the current value. R nitrif = 14 N NO 3 14 N NH + 4 (S20) (S21) R nitrifnh + 4 = R nitrif 14 N NRUM 14 N NR0 (S22) In keeping with our minimalistic approach (and the extreme uncertainties in the size/spatial distribution of the reduced nitrogen reservoir in the Proterozoic ocean) we make no attempt to develop a fully closed isotopic mass balance model - this approach is therefore merely a sensitivity analysis to δ RUM0, the initial composition of ammonium entering the upwelling zone. Clearly this factor will be impacted upon by the composition of ammonium upwelling from the deep ocean and by fractionation associated with ammonium assimilation in the photic zone. In a large reduced nitrogen pool scenario the former factor will be more relevant, and ammonium will presumably bear minimal isotopic signature (solely that from nitrogen xation). In a smaller, more modern scenario in which the deep ocean reduced nitrogen pool is smaller, enrichment in residual aqueous ammonium might conceivably occur due to preferential assimilation of the lighter isotope into organic matter in the photic zone. However in this case quantitative assimilation (and nitrication) might make the impact of ammonium on the nitrate isotopic signal trivial in any case. Either way it is dicult to constrain or develop testable predictions for these processes, and, as indicated by the sensitivity analysis below they have little impact on our qualitative arguments. The sensitivity analysis below shows the impact of an unusually large and/or isotopically unorthodox deep ocean ammonium pool on our results. Supplementary references 37. Konhauser, K. Introduction to geomicrobiology. Blackwell. pp (2007) 38. Anderson, L.A. & Sarmiento, J.L. Redeld ratios of remineralisation determined by nutrient data analysis. Global. Biogeochem. Cyc (1994) 39. Kah, L.C., Lyons, T.W. & Frank, T.D. Low marine sulphate and protracted oxygenation of the Proterozoic Biosphere. Nature (2004) 40. Schlesinger, W.H. & Hartley, A.E. A global budget for atmospheric NH 3 Biogeochemistry 15: (1992) 41. Sigman, D.M., Karsh, K.L. & Casciotti, K.L. Ocean process tracers: Nitrogen isotopes in the ocean. In Encyclopedia of Ocean Sciences (update from 2001), edited by J.H. Steele, K.K. Turekian, and S.A. Thorpe, Academic Press, London. (2009) 42. Wada, E., Kadonaga, T & Matsuo, S. abundance in nitrogen of naturally occurring substances and global assessment of denitrication from isotopic viewpoint. Geochemical Journal (1975) 19